Prevalence and concentrations of new designer stimulants, synthetic opioids, benzodiazepines, and hallucinogens in postmortem hair samples: A 13-year retrospective study.

Hair samples are frequently analyzed in order to characterize consumption patterns of drugs. However, the interpretation of new psychoactive substance (NPS) findings in hair remains difficult because of lacking data for comparison. In this study, selected postmortem hair samples (n = 1203) from 2008 to 2020 were reanalyzed for synthetic cathinones, piperazines, phenethylamines, hallucinogens, benzodiazepines and opioids to evaluate prevalence data and concentration ranges. Hair samples were extracted using a two-step extraction procedure and analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Overall NPSs were detected in 381 cases (31.6%). Many cases were tested positive for more than one NPS in the same time span. A variety of NPS with a large range of concentrations was observed. For better comparability and interpretation of positive cases in routine work, quantitation data for 13 NPS were calculated as percentiles. The most frequently detected NPS in this study were N-ethylamphetamine, α-pyrrolidinovalerophenone, mephedrone, benzedrone, metamfepramone, and 4-fluoroamphetamine. In conclusion, a high prevalence of these drugs was observed from postmortem hair samples. The results show a growing use of many different NPSs by mainly young drug-using adults. Consequently, NPS screening procedures should be included in forensic toxicology. Our quantitative data may support other toxicologists in their assessment of NPS hair concentrations.

Prevalence of cathinones and other new psychoactive substances in hair of parents and children of families with known or suspected parental abuse of conventional illegal drugs.

BACKGROUND: Hair analysis of parents and their children was regularly used since 2011 as a diagnostic tool in a social support project for families with known or suspected abuse of conventional illegal drugs and revealed a high incidence of cocaine, cannabinoids, amphetamines, ecstasy and heroin. In this context, the prevalence of new psychoactive substances (NPS) in these families should be important for a realistic estimate of the situation. METHODS: The extracts of 1537 hair samples from 318 children (age 1-14 years), 44 adolescents and 611 adults, which were collected and tested for conventional drugs between June 2016 and April 2021 and frozen at -20 °C, were reanalyzed by a validated LC-MS/MS method (limits of quantitation 5-24 pg/mg) for 33 cathinones, 10 phenylethylamines, 5 piperazines including the antidepressant trazodone, 2 tryptamines, 9 designer benzodiazepines, 4 synthetic opioids and 4 ketamine-like substances including phencyclidine. RESULTS: Between one and up to five from 42 of these substances were detected in 227 samples (14.8%). The most frequently detected substances were benzedrone (62x), α-pyrrolidinovalerophenone (41x), N-ethylamphetamine (29x), dimethyltryptamine (13x) and pyrovalerone (11x). The quantification was possible only for 34 results of 15 drugs and the remaining majority of the results were unambiguously identified below LLOQ. The relative frequency of conventional drugs in the 227 NPS positive samples was higher than in all 1310 NPS negative samples for cocaine (69.6% vs. 56.0%), heroin (6-acetylmorphine 8.8% vs. 4.9%), amphetamine (16.3% vs. 7.7%) and MDMA (16.3% vs. 7.0%) but was similar for THC (38.3% vs. 36.3%) and benzodiazepines (1.8% vs. 1.7%). The high prevalence of N-ethylamphetamine can be explained as a byproduct of the illicit amphetamine synthesis from benzaldehyde and nitroethane rather than as a separate drug or as a combined metabolite of amphetamine and ethanol. The isolated appearance of 3-trifluoromethylphenylpiperazine in 9 hair samples collected in January 2017 can be caused either by its use as an NPS or by its formation as a metabolite of the medical drug flibanserin. The results were compared within 17 families whose members were tested at the same time and showed positive NPS results. The detected drugs agreed between both parents only in about half of the cases whereas the drugs found in children's hair was always detected also in hair of one or both parents. CONCLUSION: The re-testing of hair extracts for NPS after long-time storage in frozen state enables an impression about the relative high prevalence in the tested population group, despite the limitation by partial degradation of the substances and the corresponding impossibility in quantitative assessments. In addition to conventional drugs, the hair test for these substances should be useful in unclear cases of child's welfare endangerment and in family law.

GHB related acids (dihydroxy butyric acids, glycolic acid) can help in the interpretation of post mortem GHB results.

Post mortem gamma hydroxy butyric acid (GHB) concentrations should be interpreted with caution since GHB concentrations can increase after death. Post mortem concentrations after the intake of GHB ante mortem do overlap with concentration ranges in cases without known exposure to GHB and make an interpretation challenging. GHB is known to undergo intensive metabolism to related acids (glycolic acid (GA), succinic acid (SA), 2,4- and 3,4-dihydroxy butyric acid (2,4-OH-BA and 3,4-OH-BA)). GHB and these related acids were analyzed using a validated gas chromatographic mass spectrometric (GC-MS) method after liquid liquid extraction and trimethylsilylation. SA concentrations were not usable post mortem due to instability. Concentrations in cases without known exposure to GHB (urine: n = 80; femoral blood: n = 103) were: for GA 4.6-121 mg/L in urine and 1.6-11.2 mg/L in blood, for 2,4-OH-BA < LoD-25,3 mg/L in urine and < LoD-3.7 mg/L in blood and for 3,4-OH-BA < LoD-54,3 mg/L in urine and < LoD-5.3 mg/L in blood. In death cases involving GHB (n = 11) concentrations of GHB related acids were increased compared to these levels (for GA in 7/10 cases and up to 391 mg/L in urine, in 6/11 cases and up to 34 mg/L in blood; for 2,4-OH-BA in 9/10 cases and up to 144 mg/L in urine, in 11/11 cases and up to 9.1 mg/L in blood; for 3,4-OH-BA in 7/10 cases and up to 665 mg/L in urine, in 11/11 cases and up to 19 mg/L in blood). Therefore, the concentrations of these GHB related acids can aid in a more reliable differentiation of GHB exposure in post mortem toxicology. We recommend to add the analysis of 2,4-OH-BA, 3,4-OH-BA and GA in femoral blood for the diagnosis of a GHB intake post mortem. Post mortem femoral blood concentrations > 4 mg/L for 2,4-OH-BA, > 5 mg/L for 3,4-OH-BA and > 12 mg/L for GA give hints for a GHB intake.

Hair analysis of antidepressants and antipsychotics-Overview of quantitative data.

Antidepressant and antipsychotic drugs are regularly encountered in different aspects of forensic toxicology, and some cases require the examination of hair samples. In this study, common antidepressant and antipsychotic drugs regarding hair concentrations over the past decades were reviewed. Although numerous publications around method validations, case reports, or controlled dose studies were found, apparently there is a lack of comprehensive data for many substances. Information on the hair length and dosage across the publications varied largely, and case numbers were generally low except for several retrospective controlled dose studies. Many substances were described only in method validations or case reports, and data were obtained from small case numbers. On the contrary, clozapine, haloperidol, amitriptyline, nortriptyline, risperidone and its metabolite, methylphenidate, citalopram, chlorpromazine, chlorprothixene, and quetiapine had a well-founded database as these substances were investigated in controlled dose studies with higher case numbers. Given the advancements made in analytical techniques over the past years, gas chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry techniques were the methods of choice and allowed the detection of chemical compounds at low concentrations. The controversy around a potential use of hair analysis to estimate the dosage remains as dose-concentration studies provided divergent results. A harmonization on the investigated hair length as well as on the extraction protocol would be of favor to achieve better comparability. Although hair analysis research focused mainly on drug abuse, availability of more data on antidepressants and antipsychotics would help to gain better knowledge and assist other forensic investigators.

Positive findings of ethyl glucuronide in hair of young children from families with addiction background.

AIMS: Small children are expected to be abstinent from alcohol, and children's hair is frequently used as the blank matrix for calibration of the alcohol consumption marker ethyl glucuronide (EtG). The basal EtG concentrations of total abstainers were described to be 0.3-2.1 pg/mg (Pirro et al. 2013). It is examined whether this assumption is valid for children from families with addiction background. METHODS: In a social support system for families with drug and/or alcohol addicted parents, 161 hair samples from 126 children (age 1-14 years, hair segment 0-3 cm) were analyzed for EtG by a validated LC-MS/MS method (LOD 0.56 pg/mg, LLOQ 2.3 pg/mg). For comparison, ethyl palmitate (EtPa) was measured and hair samples from parents were included. EtG ≥ 3 pg/mg was considered as an alarming result for children. RESULTS AND DISCUSSION: EtG concentrations between 3.0 and 42.6 pg/mg (mean 9.55 pg/mg, median 6.40 pg/mg) were measured for 25 samples (15.5%, age 22 × 1-5 years, 3 × 9-11 years). Elevated EtPa (0.15-0.46 ng/mg) was found in 6 samples and cocaethylene (0.02-0.07 ng/mg) was detected in 5 samples with high cocaine findings. Hair results of one or both parents indicated drug abuse in 12 from 14 cases (85.7%) if both parents were tested. CONCLUSION: Although accidental or voluntary intake of alcoholic beverages cannot be excluded, the external contamination of children's hair by EtG-containing wine and sweat or urine of the alcohol abusing parents is assumed to be the most probable explanation for the positive EtG results in hair of 1-5-year-old children.

Detection and quantification of synthetic cathinones and selected piperazines in hair by LC-MS/MS.

New psychoactive substances (NPS)-such as synthetic cathinones and piperazines-are defined as substances designed to replicate the effects of traditional illegal drugs, including cocaine, ecstasy and amphetamines. These substances are known to potentially be much more potent than their analogs. In the past, there were many poisonings and deaths associated with NPS. Because of this, NPS identification and quantification have become more important in forensic toxicology. The present work aimed to develop, validate and apply a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of detecting 35 synthetic cathinones and piperazines in hair samples. All target analytes were resolved in a 12 min run time and identified based on the quantifier ion, at least one product ion and the retention time. Depending on the analyte, the calibration curves were linear over a maximal range of 0.01-3 ng/mg. The limits of detection and quantification were within the ranges of 0.006-0.052 ng/mg and 0.008-0.095 ng/mg, respectively. The precision, bias and matrix effect were all within acceptable GTFCh thresholds and the method was free from interferences. The validated method was successfully used to identify synthetic cathinones and piperazines in authentic hair samples (n = 40) from forensic cases, demonstrating its suitability for the screening and quantification of a wide number of new stimulants in hair specimens.

Hair analysis of more than 140 families with drug consuming parents. Comparison between hair results from adults and their children.

BACKGROUND: Hair samples from children are frequently analyzed in order to characterize their endangerment in a drug using environment. However, the interpretation of the results remains difficult because of lacking data for comparison. In this study, hair samples from families with drug consuming parents were analyzed for illegal and selected medical drugs and the results were evaluated concerning a relationship between findings of parents and children depending on kind of drug, age and gender of children as well as maternal or paternal drug concentrations in hair. METHODS: In an ongoing social supporting project for families with underage children and drug consuming parents, hair samples were analyzed since 2011 for methadone, opiates and opioid analgesics, cocaine, amphetamines, ecstasy, cannabinoids and benzodiazepines by LC-MS/MS with LOQs ≈ 0.01 ng/mg. From the data pool of more than 1300 individuals, 100 families with results for one or both parents and one to five children, 30 families with results only for both parents, and 11 families with results only for 2-4 children were selected. Fifty eight of these 141 families were repeatedly tested (altogether 251 family tests). RESULTS: One to 5 drugs were detected in 239 (95.2%) of the family tests with highest occurrence of cocaine (79.7%) and THC (50.2%). According to the concentrations of the tested persons, the most probable drug users were the mother (25%), the father (24%), both parents (16%), or were not tested (30%). Within the families, there was an agreement of the detected drugs between parents and children of 47.8%, between both parents of 36.1%, and between children of 42.3%. For parents with hair concentrations in the typical range of regular drug use, the drug was detected in children hair with the following frequency: methadone 65.5%, heroin (6-AM) 63.6%, cocaine 92.1%, amphetamine 80%, MDMA 42.9% and THC 67.4% with higher percentage for younger children. The agreement for medical drugs (benzodiazepines 7.7%, synthetic opioids 8.7%, diphenhydramine 7.1%) was much lower suggesting voluntary administration or intake. Despite the strong variation of the data, clear trends were found that the child/parent drug concentration ratio decreases with increasing children age and is higher for boys than for girls. CONCLUSION: The comparison of hair results within families gives a deeper insight in the drug situation, often enables the identification of the drug user and is helpful for social and legal decisions to improve the conditions of the children.

Toxicological findings in suicides - frequency of antidepressant and antipsychotic substances.

The role of psychoactive substances in the treatment of mental disorders and the risk of suicide are major public health issues. This cross-sectional study examined the prevalence of antidepressants and antipsychotics detected in toxicological screenings in suicides. Cases from the Institute of Legal Medicine of the Charité-University Medicine Berlin were reviewed over a 4-year-period. All cases (n = 477) with positive toxicology for antidepressants and antipsychotics in blood or organ tissue were included. Frequencies of the detected substances in non-suicide cases (n = 212; male n = 177, 55.2%; female n = 95, 52.5%) and suicide cases (n = 235; male n = 149, 63.4%; female n = 86, 36.6%) were examined. Tricyclic antidepressants (48.1%) were found most frequently in suicides, followed by atypical neuroleptics (37.0%), selective serotonin reuptake inhibitors (28.1%), typical neuroleptics (17.4%), tetracyclic antidepressants (16.2%) and other substances (8.9%). Alcohol was detected in 37.2% of suicides. The leading cause of death was drug poisoning (35.6%) followed by polytrauma (26.8%) and death by hanging (18.5%). A mental disorder (depression, schizophrenia, bipolar disorder, suicidality) was known in 22.9% of suicides. The most common location of death was the person's own house (63.8%) followed by public places (28.1%) and hospitals (8.1%) The five most common substances in the suicide group were doxepin (20%) citalopram (15.3%), mirtazapine (14.9%), quetiapine (13.6%) and amitriptyline (12.3%). Toxicological findings from cross-sectional studies provide insight into how often certain types of antidepressants and antipsychotics are associated with suicide. A complementary approach is valuable for assessing the risk of suicide during medical treatment because the various available approaches (analysis of suicidal behavior/ideation, toxicity of drugs) each have strengths and limitations.

Behaviour of hygrine and cuscohygrine in illicit cocaine production establishes their use as markers for chewing coca leaves in contrast with cocaine abuse.

Hygrine (HYG) and cuscohygrine (CUS) are natural alkaloids of coca leaves but are not found in illicit cocaine seizures. Therefore, they were proposed as markers for coca chewing in contrast to cocaine abuse in urine and hair testing. In order to examine at which step of the illegal cocaine production these compounds are lost, coca leaves were processed according to an authentic procedure by extraction with lime and kerosene, re-extraction with sulphuric acid, and precipitation of coca paste with ammonia. Non-extracted and extracted coca leaves, acidic extract and coca paste were analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for cocaine, ecgonine methyl ester (EME), cinnamoylcocaine (CIN), HYG, and CUS. It follows from the results that under these conditions, HYG and CUS are extracted only to a minor extent by kerosene and are not precipitated from the acidic re-extract in the coca paste. Due to this behaviour in illegal cocaine production, they fulfil the conditions as markers for coca chewing in an optimal way. However, for unambiguous discrimination between coca chewing and cocaine abuse in human samples, additional markers of manufactured cocaine are required. Copyright © 2016 John Wiley & Sons, Ltd.

The use of nails as an alternative matrix for the long-term detection of previous drug intake: validation of sensitive UHPLC-MS/MS methods for the quantification of 76 substances and comparison of analytical results for drugs in nail and hair samples.

PURPOSE: This article describes the validation of multi-target methods for the determination of 76 different analytes in hair and nail samples. Segmented hair and nail samples taken from autopsy cases were included in this study. METHOD: Drugs of abuse, psychotropic drugs, and other drugs were included for the validation of this method. Hair and nail samples were ground using a ball mill and extracted for 18 h. Extracts were measured using a UHPLC-triple quadrupole-mass analyzer. Analytes were separated on a RP 18 column under gradient elution of the mobile phases, water with 0.1 % formic acid and acetonitrile. Whole nail and hair samples from seven autopsy cases were split into segments and analyzed. RESULTS: The entire method was validated according to the German Society of Forensic Toxicology guidelines. In addition, the concentration ratios of selected substances and their metabolites were calculated. Similar concentration ratios in hair and nails were detected for 3,4-methylendioxyamphetamine (MDA)/3,4-methylenedioxy-N-methylamphetamine (MDMA), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)/methadone, and bisnortilidine/nortilidine in some of the cases. Reduced 6-monoacetylmorphine (6-MAM) and cocaine concentrations were observed as a result of sample preparation using the ball mill. Previous heroin intake could be assumed from the detection of 6-MAM and morphine in only one nail segment in one case. CONCLUSIONS: Nail samples may serve as an alternative matrix for the detection of long-term consumption of a wide range of drugs. Based on our results, drug concentrations in nails are not comparable to those in hair. The main mechanisms for drug incorporation into the nails may be during the formation of the nail plate by the germinal matrix. However, external contamination can also affect the analysis of nail clippings.

Nails are a potential alternative matrix to hair for drug analysis in general unknown screenings by liquid-chromatography quadrupole time-of-flight mass spectrometry.

PURPOSE: In this study, the usefulness of nail samples instead of hair for a general unknown screening (GUS) for drugs was tested. An alternative matrix for long term detection is still needed in cases where no hair is available for analysis. METHODS: Hair and nail samples from 70 postmortem cases were analyzed by liquid-chromatography quadrupole time-of-flight mass spectrometry. Hair and nail samples were ground by a ball mill and extracted twice for 18 h. Extracts were measured in Auto MS/MS mode (data dependent mode acquisition). RESULTS: Only 10% of the cases showed a disagreement of results in hair and nail analysis where hair samples were tested positive and corresponding nail samples were tested negative in a general unknown screening for drugs. In most of the cases investigated the analysis of the nail clippings and whole nail samples led to results comparable to those obtained from hair analysis. The incorporation of a large number of substances into the nail matrix was proven by the detection of 89 different analytes (e.g. antidepressants, drugs of abuse or antihypertonics) in our tests. CONCLUSION: In cases where the amount of hair available is not sufficient for a general unknown screening for drugs, nails appear to be a useful comparable matrix for the detection of long-term drug consumption due to the comparison of the qualitative GUS results from the hair and nail samples in this study.

Hair analysis in the detection of long-term use of non-steroidal anti-inflammatory drugs and its relation to gastrointestinal hemorrhage: an examination of 268 hair and blood samples from autopsy cases.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used as analgesics and antipyretics in Western countries. Gastrointestinal (GI) disorders are common side effects of NSAIDs and other drugs. This study investigated the correlation between chronic use of these substances and GI lesions by analyzing postmortem blood and hair samples from autopsy cases. This study included 268 hair and blood samples from autopsy cases. Deceased individuals with GI lesions were selected for the case group (n = 132) and those without any GI lesions were placed in the control group (n = 136). Collection of the samples took place from 2008 until 2010 at the Institute of Legal Medicine and Forensic Sciences, Charité-Universitätsmedizin Berlin, Germany. HPLC-DAD was used to analyze the blood samples while hair samples were analyzed using LC-quadrupole-time-of-flight-MS. The proximal 0-6 cm hair segment was analyzed. The full length of shorter hair samples was analyzed when longer segments were unavailable. Method validation was performed according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Twenty-three per cent of the case group blood samples included one or more NSAIDs while 19 % of the control group blood samples included one or more NSAIDs. In contrast, the hair analysis results demonstrated that samples from the control and case group differed significantly; 67 % of the case group tested positive for one or more NSAIDs while 38 % of the control group tested positive for one or more NSAIDs. Hair analysis results provided a strong indication of a relationship between frequent NSAID consumption and GI lesions.

Fatty acid ethyl esters in hair as alcohol markers: estimating a reliable cut-off point by evaluation of 1,057 autopsy cases.

Alcohol abuse is a widespread problem, especially in Western countries. Therefore, it is important to have markers of alcohol consumption with validated cut-off points. For many years research has focused on analysis of hair for alcohol markers, but data on the performance and reliability of cut-off values are still lacking. Evaluating 1,057 cases from 2005 to 2011, included a large sample group for the estimation of an applicable cut-off value when compared to earlier studies on fatty acid ethyl esters (FAEEs) in hair. The FAEEs concentrations in hair, police investigation reports, medical history, and the macroscopic and microscopic alcohol-typical results from autopsy, such as liver, pancreas, and cardiac findings, were taken into account in this study. In 80.2 % of all 1,057 cases pathologic findings that may be related to alcohol abuse were reported. The cases were divided into social drinkers (n = 168), alcohol abusers (n = 502), and cases without information on alcohol use. The median FAEEs concentration in the group of social drinkers was 0.302 ng/mg (range 0.008-14.3 ng/mg). In the group of alcohol abusers a median of 1.346 ng/mg (range 0.010-83.7 ng/mg) was found. Before June 2009 the hair FAEEs test was routinely applied to a proximal hair segment of 0-6 cm, changing to a routinely investigated hair length of 3 cm after 2009, as proposed by the Society of Hair Testing (SoHT). The method showed significant differences between the groups of social drinkers and alcoholics, leading to an improvement in the postmortem detection of alcohol abuse. Nevertheless, the performance of the method was rather poor, with an area under the curve calculated from receiver operating characteristic (ROC curve AUC) of 0.745. The optimum cut-off value for differentiation between social and chronic excessive drinking calculated for hair FAEEs was 1.08 ng/mg, with a sensitivity of 56 % and a specificity of 80 %. In relation to the "Consensus on Alcohol Markers 2012" by the SoHT, an increase in the cut-off value for FAEEs in the proximal hair segment 0-3 cm from 0.5 to 1 ng/mg may be advisable to avoid excessive numbers of false positive results.

Detecting alcohol abuse: traditional blood alcohol markers compared to ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) measurement in hair.

Alcohol abuse is a common problem in society; however, the technical capabilities of evaluating individual alcohol consumption using objective biomarkers are rather limited at present. In recent years research has focused on alcohol markers using hair analysis but data on performance and reliable cut-off values are still lacking. In this study 169 candidates were tested to compare traditional biomarkers, such as carbohydrate-deficient-transferrin (CDT), gamma glutamyl transferase (GGT), aspartate amino transferase, alanine amino transferase and the mean corpuscular volume of the erythrocytes, with alcohol markers detectable in hair such as ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). This study revealed that EtG, GGT and CDT showed the best results, demonstrating areas under the curve calculated from receiver operating characteristics of 0.941, 0.943 and 0.899 respectively. The lowest false-negative and false-positive rates were obtained by using a combined interpretation system for hair EtG and FAEEs. All markers demonstrated only low to moderate correlations. Optimum cut-off values for differentiation between social and chronic excessive drinking calculated for hair EtG and FAEEs were 28 pg/mg and 0.675 ng/mg, respectively. The critical values published in the "Consensus on Alcohol Markers 2012" by the Society of Hair Testing were confirmed.

Fatty acid ethyl esters (FAEEs) as markers for alcohol in meconium: method validation and implementation of a screening program for prenatal drug exposure.

Alcohol consumption during pregnancy is a widespread problem and can cause severe fetal damage. As the diagnosis of fetal alcohol syndrome is difficult, the implementation of a reliable marker for alcohol consumption during pregnancy into meconium drug screening programs would be invaluable. A previously published gas chromatography mass spectrometry method for the detection of fatty acid ethyl esters (FAEEs) as alcohol markers in meconium was optimized and newly validated for a sample size of 50 mg. This method was applied to 122 cases from a drug-using population. The meconium samples were also tested for common drugs of abuse. In 73 % of the cases, one or more drugs were found. Twenty percent of the samples tested positive for FAEEs at levels indicating significant alcohol exposure. Consequently, alcohol was found to be the third most frequently abused substance within the study group. This re-validated method provides an increase in testing sensitivity, is reliable and easily applicable as part of a drug screening program. It can be used as a non-invasive tool to detect high alcohol consumption in the last trimester of pregnancy. The introduction of FAEEs testing in meconium screening was found to be of particular use in a drug-using population.